This research proposal is aimed to elucidate the molecular and genetic mechanisms of Drosophila hematopoiesis. Special attention will be given to Toll/Rel signaling, which is equivalent to the mammalian counter part, IL1 -R/NFkappaB signaling. Both signal transduction pathways play a role in the innate immunity, an evolutionarily conserved defense mechanism. Moreover, most molecules in these pathways are conserved between Drosophila and mammals. Interestingly, hyperactivation of Toll/Rel signaling by mutations results in leukemia-like symptoms, which is a hallmark in this system. The major goals of this proposal are to understand the genetic and molecular mechanisms of Cactus (Drosophila IkappaB homologue) degradation and to identify hematopoietic genes by mutagenesis as well as microarray expression profiling. While many Drosophila mutations in Toll/Rel signaling will be used in this proposal, the lesswright (lwr) gene is very unique among them. lwr encodes a Drosophila Ubc9 homologue, which is similar in sequence to ubiquitin-conjugating (E2) enzymes, however Ubc9 does not participate ubiquitination. Rather, they conjugate small ubiquitin-like modifiers (SUMO) instead of ubiquitin. Ubiquitin and SUMO possess similar biochemical properties such that conjugation of both of these molecules occurs at a lysine residue on a target protein. In the case of Cactus (Cact), their target residues are at identical positions. Thus, ubiquitination and SUMO-conjugation counteract each other for Cact protein degradation, which is a crucial regulatory step for activation of Rel-family transcription factors, Dif (dorsal related immunity factor) and Dorsal (Dl). This is a unique situation where Cact degradation does not solely depend on a stimulatory signal input from the Toll receptor. Genetic and biochemical approaches will be taken to investigate mechanisms of Cact degradation. Mutagenesis will be conducted to isolate mutations that enhance or suppress lwr mutant phenotypes (overproduction of blood cells in larva). Some will be mutations of hematopoietic genes. Microarray analysis will be performed to identify additional hematopoietic genes in Toll/Rel signaling. Our first analysis has identified several potential genes, thus another series of analyses will be conducted to make a comprehensive list of hematopoietic genes. Thus, the outcome of this proposed research will be highly significant and contribute to a general understanding of hematopoiesis including humans.